Cavity-Creating Mutations in Pseudomonas aeruginosa Azurin: Effects on Protein Dynamics and Stability

Abstract

Changes in flexibility and structural stability of Pseudomonas aeruginosa azurin in response to cavity-creating mutations were probed by the phosphorescence emission of Trp-48, which was deeply buried in the compact hydrophobic core of the macromolecule, and by measurements of guanidinum hydrochloride unfolding, respectively. Replacement of the bulky side chains Phe-110, Phe-29, and Tyr-108 with the smaller Ala introduced cavities at different distances from the hydrophobic core. The phosphorescence lifetime (tau(0)) of Trp-48, buried inside the protein core, and the acrylamide quenching rate constant (k(q)) were used to monitor local and global flexibility changes induced by the introduction of the cavity. The results of this work demonstrate the following: 1), the effect on core flexibility of the insertion of cavities is not correlated readily to the distance of the cavity from the core; 2), the protein global flexibility results are related to the cavity distance from the packed core of the macromolecule; and 3), the increase in protein flexibility does not correspond necessarily to a comparable destabilizing effect of some mutations.

Subunit interfaces of oligomeric hyperthermophilic enzymes display enhanced compactness

“…The importance of compactness in protein stability and thermostability has been supported by several theoretical and experimental studies [30][31][32]. In our dataset, the average distance between occluding atoms from the opposite subunits of the oligomer turned out to be significantly lower in the hyperthermophilic proteins than in the mesophilic counterparts [10].…”

Beta-Barrel Scaffold of Fluorescent Proteins

“…At the same time, the rate of unfolding of pOBP is 1000 times faster than that of the GFP-like proteins under the same conditions (Staiano et al, 2007). Interestingly, azurin, which has a single tryptophan residue (Trp48) deeply buried in the hydrophobic central cavity of the β-barrel and thus resembles the FPs, is also much less stable to denaturant action (Gabellieri et al, 2008). …”

Tuning Structure and Dynamics of Blue Copper Azurin Junctions via Single Amino-Acid Mutations

“…Interestingly, several reports [20,21] show that point-like mutations which retain the protein’s original structure strongly affect its flexibility/vibrations, in some cases almost doubling its stiffness [21,22]. These observations are general as they hold not only for the Azurin [20,23,24,25], but also in many other proteins [26,27,28,29,30,31]. Thus, tuning protein dynamics via point-like mutations may offer yet another way of controlling their adsorption dynamics and configurations.…”